The invention concerns a method for the supply of fluid volume streams in channels or capillaries with small stream cross sections, more particularly in chromatographic separation columns for analytical fluid metrology, more particularly for analytical liquid chromatography, wherein a delivery device for the delivery of a volume stream of the fluid through an operating channel and a pressure measuring device for measuring the pressure in the operating channel are provided, wherein a measurement of the volume stream is possible.
Such a method is presently used, for example, in liquid chromatography, more particularly high pressure liquid chromatography (HPLC). Depending on the inner diameter of the separating columns used, HPLC is divided into so-called xe2x80x9cnormal bore chromatographyxe2x80x9d, in the case of separating columns with an inner diameter between approximately 3 and 5 mm, xe2x80x9cmicro bore chromatographyxe2x80x9d, using separating columns with an inner diameter between approximately 1 and 2 mm, xe2x80x9ccapillary-LC-chromatographyxe2x80x9d using separating columns with an inner diameter between approximately 180 and 320 xcexcm, and xe2x80x9cnano-LC chromatographyxe2x80x9d, using separating columns with an inner diameter equal to or smaller than 100 xcexcm.
The supplied flow rates must be adapted to the inner diameter of the column according to the application. While flow rates in the range of ml/min and xcexcm/min are common in normal bore technology and micro bore technology, flow rates in the range of as low as several 100 nl/min must be realized in nano-LC technology. The flow rates are commonly adjusted so that in the separating column a linear stream velocity of approximately 1 to 2 mm/s is achieved. This is important because the efficiency of a separating column depends on the flow rate.
The application of separating columns with an inner diameter of less than 180 xcexcm is gaining interest not only in high pressure liquid chromatography but also in the area of other microfluid systems. While in high pressure liquid chromatography the volume stream is commonly generated using a hydraulic pump, electric osmosis is often used to create the volume streams in microfluid systems. Evidently, it is also possible to combine hydraulically pumps with microfluid systems. A particular example of such a microfluid system is a microfluid chip.
Another important point in the connection with an optimum flow rate is the detection of the substances to be analyzed. Mass-selective detectors are increasingly being used. The use of such detectors requires suitable preparation and supply of the substances to be analyzed. For this purpose, the operating stream can be atomized, ionized and partially or completely dried after passing through the separating column. The individual ions stream into the opening of the detector while the solvent excess that might still exist is waste. Various methods are known for the creation of the ions. Some methods only work in a certain range of flow rates or volume flow rates. For lower or higher flow rates, the method does not work or works with significant restrictions, i.e. the detection is less sensitive or impossible.
The lower the flow rates, the more important the effects of system volumes, in particular the dead volume or the delay volume. In the case that is of interest here, the case with extremely low flow rates, these volumes must also be flushed with a very low flow rate. Otherwise, thermodynamic effects could disturb the equilibrium of the separating column and of the detector so that undesired effects may occur. Also, high flow rates are impossible simply because of the connection capillaries with very small internal diameters and the consequent pressure drop. To ensure a high efficiency of the analysis, the volumes mentioned above should be kept as small as possible. Ideally, a flush period of one minute should be sufficient to flush the delay volume with the desired low flow rate.
In a chromatographic system, the parameter stream and pressure are always interdependent via the hydraulic resistance of the separating column and the system. However, it has become common practice to determine or define the flow. The linear stream velocity through the column must be kept constant regardless of the transported solvents and the restrictions. This does not pose a problem as long as the flow rates are high enough to either directly supply the desired flow rate or to measure the flow rate. However, if extremely low flow rates in the range of nl/min are to be supplied, the two above-mentioned methods can normally not be applied.
For the above-mentioned applications, pump systems are required that can create or transport extremely low flow rates or volume streams. The delivery must be highly reliable at the high existing pressures in the range of approx. 400 bar.
For the delivery and provision of such small flow rates in separating columns for liquid chromatography, in particular for xe2x80x9ccapillary LC chromatographyxe2x80x9d and for xe2x80x9cnano-LC chromatographyxe2x80x9d, the only methods currently known are as follows:
A first method is based on the application of so-called syringe pumps. Syringe pumps are special one-piston pumps. Unlike in common piston pumps, the pistons do not move back and forth during the analysis. Instead only one piston stroke is performed. This means that the syringe pumps always work in delivery mode. The pump chamber must therefore be chosen sufficiently large so that a single piston stroke is sufficient for a complete separating analysis. The pump chamber is put under pressure before the analysis by pushing the piston in the pump chamber forward. No further suction is thus performed during the separating analysis. With this method, a volume flow can be achieved that is independent of the elasticities inside the pump chamber. The elasticities of particularly the seals and the drive mechanics as well as the elasticity due to the compressibility of the solvent can be compensated for accordingly.
However, syringe pump technology has little flexibility in the realization of different analysis times and in the use of different column diameters. This is because the possible analysis time and the choice of the separating column diameter is dependent of, and limited by, the respective available maximum displacement volume of the syringe pumps. In addition, only one high pressure gradient can be realized with a syringe pump. This means that a separate high-pressure syringe pump is necessary for every solvent used in the analysis.
Further more, the leak rate inside the delivery system plays an important role for the delivery amounts of only several nl/min. The seals and valves used for high-pressure syringe pumps typically have relatively low leak rates. Nevertheless, these leak rates can have a dramatic effect for the small flow rates used in nano-LC chromatography. Even temperature influences can cause undesired flow shifts due to thermal extension. For this reason, special thermostat arrangements and controls are often necessary.
Another possibility for the creation and provision of liquid volume streams in channels or capillaries with small diameters is the use of traditional piston pumps suitable for xe2x80x9cnormal bore chromatographyxe2x80x9d. This method uses the so-called passive splitter technology, which is very common in practice. It means that suitable flow divider are used to divide the total stream created and supplied by the pump into at least two partial streams, a surplus stream in a surplus path and an operating stream in an operating path.
The regulation and provision of the respective operating stream is done by so-called restrictors, i.e. by hydraulic resistances located in the surplus path. The flow dividers, and in particular the hydraulic resistors, are usually made of so-called xe2x80x9cfused silica capillariesxe2x80x9d with small inner diameter. The length and the inner diameter of these elements determine the stream resistance. The total flow rate is split according to the resistance ratios. Typically, the smaller part flows through the separating column.
The advantage of this technology is the low production expenditure because the splitters and the hydraulic resistances can be produced by the users themselves. The extremely low volumes inside the flow dividers or inside the hydraulic resistances are also of advantage.
A particular disadvantage of the traditional splitter technology, however, is the fact that the users do not receive any information on the volume streams that flow through the separating column. For this reason, the volume stream must be measured at great effort, for example using miniature syringes and stop watches, to operate the separating column efficiently. Furthermore, even minute changes in the flow resistance, caused for example by dirt in the separating column frits, cause a considerable variation in the column stream. Not only the separating column, but also and in particular the restriction capillaries used for the splitter, with a diameter of 25 xcexcm for example, have a high risk of getting plugged. Such plugs consequently cause an accordingly large retention time shift. Even more serious is the effect that the reduced column volume stream can have on the above-mentioned mass-selective detectors because, as also previously mentioned, only a small dynamic stream range is available for a sensitive detection.
In order to somewhat reduce this effect, a hydraulic pre-resistance is sometimes inserted before the splitter. This causes the effect of a plugged separating column frit on the column stream to be approximately halved while keeping the pressure drops over the separating column and the pre-resistance constant. However, the use of such pre-resistances also means that only half the pump pressure is available for the separating analysis in the separating column.
Another disadvantage of the splitter technology is that the two volumes of the operating path, including the column and the surplus path, must be adjusted to each other, which for practical reasons is not normally done. The xe2x80x9cmatchingxe2x80x9d of the volumes is therefore important so that the splitter is filled uniformly in case of time variation of the flow properties of the delivered fluid, in particular the composition or concentrations of the fluid changing over time as it is always the case during gradient operation. Otherwise, this causes an additional shift of the ratio between operating stream and surplus stream because the viscosities of the fluids in the stream branches differ. In practical applications this means that for different column dimensions, the splitters must always be adjusted as well.
From DE 199 14 358 A1, an active splitter system is known with which the operating stream can be measured and kept constant. For this purpose, a suitable sensor is located in the operating stream branch and there is variable restriction in the surplus stream branch, wherein these elements are coupled with a control device, creating a control loop.
An advantage of this technology is that any common pump for chromatography can be used and the desired controlled operating stream can be branched off. In this manner, an essentially constant operating stream can be created independently of the hydraulic resistance.
However, it is difficult to perform volumetric measurements of extremely low flow rates of only several nl/min. In addition, the sensors located in the operating stream create an additional delay volume. Additional volume, on the other hand, means longer analysis time as well as stretched or shifted gradient profiles causing lower efficiency of the separating column.
Another method is based on the delivery of liquids, using essentially constant pressure. With this method a certain pressure is determined so that when the application begins, the fluid can be delivered with the desired flow rate, i.e. the desired volume stream. The pressure is essentially kept constant over the entire analysis time.
An advantage of this method is that the pressure can be measured and controlled very well. The stream that eventually flows through the column is not taken into consideration here. The delay volume of the operating branch or path can also be kept to a minimum because it is not necessary to have a sensor element in the operating stream. The pressure is commonly measured in the total stream.
The main disadvantage of this technique is the fact that the flow rate in the operating stream must first be determined by some method. For this purpose, a micro-liter syringe is connected to the exit manually and the delivered flow rate is measured volumetrically. This procedure is very work-intensive.
Furthermore, in case of a change in the flow properties, in particular the viscosity of the fluid over time or the hydraulic resistance over time, the flow rate changes and this effect is not recognized or taken into consideration. In particular in the gradient mode, the flow rate will always change due to the time-varying viscosity of the fluid when this method is applied. Gradient operation means that a concentration profile, preferably a linear concentration profile, of solvents is purposely set and delivered over time. Usually a transition from watery solutions to organic solutions takes place.
Especially for mass-selective detectors in combination with the ionization sources used in this detectors, the constancy of the xe2x80x9cresponsexe2x80x9d of the detector depends on the current flow rate. Since the flow rate is shifted significantly during the gradient operation for methods relying entirely on pressure control, the sprayer must deliver a constant spray over a larger stream area. This requires especially large effort for so-called nano-electric sprayers for very low flow rates.
It is therefore an object of the invention to provide a method such as the above-mentioned methods with which in particular fluids with different flow properties can be delivered at essentially constant flow rates without requiring these very small flow rates to be measured directly.
This object is achieved according to a first approach of this invention according to the characteristics of claim 1 by providing the following steps:
a) Recording a time reference pressure course using the pressure measuring device in the operating channel, while the delivery device delivers preferably an essentially constant, pre-determinable reference volume stream through the operating channel of a fluid with properties, more particularly flow properties, varying over time;
b) Calculation of a time-dependent operating pressure course, which corresponds to a preferably essentially constant operating volume stream through the operating channel, wherein this stream is different from, and preferably smaller than, the reference volume stream, and wherein the operating volume stream is desired for a subsequent separating analysis or substance analysis, and wherein this calculation is performed using a pre-determinable mathematical algorithm;
c) Delivery of a fluid through the operating channel, wherein the fluid preferably has the same flow properties over time as the fluid used in step a), using the time development of the operating pressure calculated in step b), while the desired work process, more particularly the separation of substance analysis, is performed.
With these measures, even extremely low flow rates, such as the flow rates in nano-LC technology in chromatographic separating columns or in microfluid systems, more particularly microfluid chips, can be delivered reproducibly and monitored or controlled. For the delivery by the delivery device, it is irrelevant whether the operating stream is created directly or is only a partial stream of a total stream. Only one device is necessary to record the pressure that occurs for a given flow rate. Lastly, there needs to be a possibility for determining the flow rate. This can be done manually or, preferably automatically.
If the measurement of the reference volume stream is performed by one or more sensors, it is preferably irrelevant at which exact positions in the system the sensors are located. Their positions can then be freely chosen according to the needs of the user. For example, the stream monitoring can be performed at the end, i.e. after the separating column in direction of the flow. In this manner it can advantageously be avoided that the sensor creates an additional delay volume, which would cause the above-mentioned disadvantages.
The invention takes advantage of the connection between pressure and flow rate in a microfluid system, more particularly a chromatographic separating system. To perform the method according to the invention, it is useful if a control device for controlling the pressure in the operating channel and a control device for controlling the volume stream in the operating channel are used and can be operated alternatively so that in step a), the reference volume stream is kept essentially constant and so that in step c), the operating pressure is kept essentially constant.
As described above, a volume-controlled delivery is certainly desired. The most important advantages are the maximum efficiency of the microfluid system, more particularly a maximum separating rate of the column and optimized spray conditions for the entry into a mass-selective detector.
The algorithm to be applied can be determined for example by performing one or more reference measurements, using one fluid at a time, which has the same flow properties varying over time, i.e. the same time-dependent flow properties course. For these reference measurements, a different but sufficiently large reference volume stream is set each time so that it can be measured with sufficient accuracy using the available measurement methods. For the direct flow measurement, a mass flowmeter or a volumetric flowmeter is particularly suitable. Such a direct measurement of the volume stream can be performed by one or several suitable sensors. On the basis of the volume stream measurement, a suitable control device can be used to control the volume stream in a control loop such that it is essentially constant over time.
It is useful if the fluid with the time-varying flow properties is a fluid gradient. The gradient is advantageously created by mixing two liquids, preferably water and an organic solution, more particularly acetonitrile, in concentrations changing over time and by transporting the fluid mixture to the separating column. Advantageously, the concentration of water is reduced linearly while at the same time the concentration of the organic solution is increased.
It is advantageous to use the following equation as the mathematical algorithm:
PA=PRxc2x7kxc2x7VA/VR 
where PA is the operating pressure, PR is the reference pressure, k is a variable or a factor, VR is the operating volume stream, and VR is the reference volume stream.
It is also advantageous if the factor 1 is used for k in the mathematical algorithm. This is of particular advantage for a chromatographic separating system in which, during the analysis or substance separation in the separating column, a linear flow velocity of approx 1 to 5 mm/s is desired for laminar flow.
According to an alternative approach of the invention, a method of this type with the previously described characteristics can have the following steps:
a) Recording a reference time development of the pressure, using the pressure measuring device in a operating channel, used as a reference channel, wherein this channel has a reference stream cross section, while the delivery device delivers a preferably essentially constant and predeterminable reference volume stream through the operating channel of a fluid with properties, more particularly flow properties, varying over time;
b) Delivery of a fluid through the operating channel, wherein the fluid preferably has the same flow properties over time as the fluid used in step a), using the time development of the operating pressure that corresponds to the time reference pressure development determined in step a), wherein the operating channel has an operating cross-section that is different from the reference cross-section of the reference channel.
For this approach it is useful to choose the reference volume stream such that there is an essentially constant and linear flow velocity in the reference channel, which is useful for the execution of the second step, i.e. the actual work process of the analysis. For chromatographic separating columns, this linear flow velocity is chosen, dependent on the particle diameter of the packing material, in the range of 1 to 5 mm/s, wherein the corresponding reference volume stream can be measured experimentally or calculated with a known equation.
In the second step, the operating pressure course over time is followed as in the first step so that the same linear flow velocity as in the first step is created in the operating channel, which has a very small operating flow cross-section, such as the operating channels used in chromatographic separating columns for LC technology. This is concluded from the realization that in channels with different cross-sections the same linear flow velocity occurs when the same pressure profile is applied.
With this method according to the second approach, the same advantage can basically be achieved as in the method according to the first approach. An additional advantage of the second approach is that the intermediate step of calculating a transformed pressure course can be omitted. However, this method requires the use of two channels with different flow cross-section, more particularly two capillaries with different diameter, whereas in the first approach, the reference measurement and the work application can be performed in the same operating channel.
Both alternative procedures have the advantage that the process can be performed directly inside the operating channel so that the existing boundary conditions in the present operating system are taken into account. These boundary conditions cannot be determined with theoretical predictions or only with extremely high effort.
It is useful to use the same operating channel or separating column for the steps a) and c) of the first method according to claim 1. However, it is also possible to use a different operating channel or a different separating column for the two steps, as it is the case for the second method according to claim 4. In these cases, it is useful if, for the steps a) and c) according to claim 1 or for the steps a) and b) according to claim 4, operating channels with essentially the same hydraulic resistances are used. When using chromatographic separating columns, it is also useful if they have essentially the same length and if their packages contain particles with essentially the same particle property, more particularly particle size, particle size distribution and/or porosity. With these measures, the use of additional corrections or correction parameter can be avoided. For this purpose, it is useful if the hydraulic resistances or restrictions of the operating channels or separating columns are essentially the same for identical linear flow velocities. This avoids different gradient selectivities.
It is useful for both methods to take into account a delay volume that may exist in the operating channel, more particularly a delay volume between a mixing point where the fluid with time-varying flow properties is created and the separating column 23, in such a manner that the time of the application of the time development of the operating pressure is chosen with a certain difference delay time as correction. Consequently, a delay time correction is performed in this manner, which appears useful in case of existing delay volumes to obtain even more precise results.
It is also useful if in the application of the essentially constant reference volume stream, a reference delay time is determined for the fluid, for example by an experiment, and then the delay volume is calculated as the product of reference delay time and reference volume stream, a work delay time is calculated as the quotient of the delay volume and the chosen operating volume stream, and the difference delay volume is calculated as the absolute value of the difference between the operating delay time and the reference delay time. The operating pressure courses can then later be applied to the reference pressure courses shifted by this difference delay time. In other words, the operating pressure courses are simply shifted by the difference delay time while maintaining their relative time development. It is useful to work with relatively high delivery volume streams, which allow for a sufficiently precise measurement in the respective reference channels while, during the actual work step or work process, only extremely low volume streams are delivered through the operating channels with very small flow cross-sections. For this reason, the time of the start of the application of the respective time development of the pressure is moved back by correspondingly longer times during the work process of the separating analysis. In this manner, the time is taken into consideration that the fluid needs to overcome the delay volume when applying the time development of the operating pressure. The separating analysis can thus be performed with the extremely low flow rates or volume streams with maximum precision.
Other advantages, characteristics, and aspects of the invention can be seen from the following description section that describes two preferred embodiments of the invention using the figures.